4 5) REAGENTS AND SAMPLE PREPARATION We recommend EDTA-plasma as sample type because in serum a time dependent increase in peroxide concentration is observed. When serum is chosen, please make sure that during preparation of serum a no longer time period than 30 min at room temperature is allowed for clotting. Store EDTA or serum samples at 20 C. Heparinized plasma should not be used for this assay, because it often precipitates upon storage and thus changes the results. Heparin-plasma, lipemic or haemolytic samples may give erroneous results. Cloudy samples should be centrifuged at least 5 minutes at 5000xg before used in the assay. All samples should be mixed well before assaying. Reconstitute as follows: Dissolve one CAL (calibrator) and each CTRL (control) in 250 µl solution D and leave at room temperature (18-26 C) for five minutes, mix well. Reconstituted CAL and CTRL can be stored aliquoted and frozen at -20 C up to one month. The following volume of ABC-reaction-mix is sufficient for 40 tests (wells) and should be used as guidance. However, the volume should be adjusted to the respective number of samples. Prepare the ABC-reaction-mix immediately before the assay by mixing: 5 ml solution A µl solution B + 5 µl solution C 6) PRINCIPLE OF THE ASSAY The peroxide concentration is determined by reaction of the biological peroxides with peroxidase and a subsequent color-reaction using TMB as substrate. After addition of a stop solution, the coloured liquid is measured photometrically at 450 nm. A calibrator is used to calculate the concentration of circulating biological peroxides in the sample (one-point calibration) 7) ASSAY PROTOCOL All reagents and samples must be at room temperature (18-26 C) before use in the assay Mark position for CTRL/CAL/SAMPLE (Control/Calibrator/Sample) on the protocol sheet Take microtiterstrips out of the plastic bag and mark as appropriate. Store unused strips in the bag.. Add 10 μl CTRL/CAL/SAMPLE (Control/Calibrator/Sample) into respective well Add 100 μl SOLA (Solution A Sample buffer) into each well Measurement 1: Determine adsorption with an Elisa reader at 450 nm Add 100 µl of the freshly prepared ABC-reaction-mix into all wells (see section 5) Reagents and Sample preparation). Incubate 15 minutes at 37 C Add 50 µl STOP (Stop solution) into each well Measurement 2: Determine absorption with an ELISA reader at 450 nm Assay procedure for automated pipetting systems Adjust volumes of the reaction solutions and samples according to the analyser used. If the analyzer is incapable of adding the stop-solution, perform kinetic measurements at 405 nm or at nm without stopping the reaction. Measurement time: 15 minutes at C. 4/20

5 8) CALCULATION OF RESULTS The differences between measurement 1 and 2 are proportional to the peroxide concentrations of the samples. For each CTRL/CAL/SAMPLE (Control/Calibrator/Sample) subtract the OD-values of measurement 1 from the OD-values of measurement 2 (= OD) A single-point calibration is done using the CAL (Calibrator). The OD-value is proportional to its concentration, which is stated on the label. This concentration is stated as H2O2-equivalents (µmol/l). The concentrations of controls and samples are calculated according to the following formula: [ mol / l] sample OD sample [ mol / l] calibrator OD calibrator 9) ASSAY CHARACTERISTICS Normal range: Linearity: Sample volume: Incubation time: Detection Limit: Reference values from apparently healthy persons with no documented disease and medication: EDTA-plasma: <400 µmol/l Serum: <350 µmol/l Median: 372 µmol/l Each laboratory should establish own normal values Up to 600 µmol/l l 10 µl human EDTA plasma, Serum and other Biological fluids 15 min 7 µmol/l 10) PRECISION Intra-Assay (n = 12) Inter-Assay (n = 12) Mean (µmol/l) 221 µmol/l Mean (µmol/l) 221 µmol/l SD 6.9 µmol/l SD 11.3 µmol/l CV% 3.1% CV% 5.1% 11) TECHNICAL HINTS Do not mix or substitute reagents with those from other lots or sources. Do not mix stoppers and caps from different reagents or use reagents between lots Do not use reagents beyond expiration date. Protect reagents from direct sunlight. Avoid foaming when mixing reagents Stop solution should be added to the plate in the same order as the reaction-mix solution 5/20

6 12) PRECAUTIONS All test components of human source were tested with 3rd generation tests against HIV-Ab and HBsAg; and were found negative. Nevertheless, they should be handled and disposed as if they were infectious. All liquid reagents contain 0.01%Proclin 300 as preservative. Avoid contact with skin and mucous membrane. Proclin 300 is not toxic in concentrations used in this kit. It may cause allergic skin reactions-avoid contact with skin or eyes. Do not pipette by mouth. Do not eat, drink, smoke or apply cosmetics where reagents are used. Avoid all contact with the reagents by using gloves. Sulfuric acid is irritating to eyes and skin. Flush with water if contact occurs. Avoid contact with skin and mucous. Irritations are possible - Flush with water after contact!! 13) LITERATURE Montine et al., American Journal of Pathology (1999) 155: Roozendaal et al., Clin. Exp. Immunol. (1999) 116: Smolle K.H. et al., Abstracts of the 7th Vienna Shock Forum (1999) Reifenbach et al. Anitoxidants & Redox Signaling, 4, Nr p Hildebrandt W, et al. Blood 2002; 99; Schimke I, et al. J. Am. CollCardiol. 2001; 38; /20

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